Fusion proteins of mycobacterium tuberculosis antigens and their uses

ABSTRACT

The present invention relates to fusion proteins containing at least two  Mycobacterium tuberculosis  antigens. In particular, it relates to bi-fusion proteins which contain two individual  M. tuberculosis  antigens, tri-fusion proteins which contain three  M. tuberculosis  antigens, tetra-fusion proteins which contain four  M. tuberculosis  antigens, and penta-fusion proteins which contain five  M. tuberculosis  antigens, and methods for their use in the diagnosis, treatment and prevention of tuberculosis infection.

The present application is a continuation-in-part of co-pendingapplication Ser. No. 09/223,040 filed Dec. 30, 1998, and of co-pendingapplication Ser. No. 09/056,556 filed Apr. 7, 1998, which is acontinuation-in-part of co-pending application Ser. No. 09/025,197 filedFeb. 18, 1998, which is a continuation-in-part of co-pending applicationSer. No. 08/942,578 filed Oct. 1, 1997, which is a continuation-in-partof co-pending application Ser. No. 08/818,112, filed Mar. 13, 1997, eachof which is incorporated by reference herein in its entirety.

1. INTRODUCTION

The present invention relates to fusion proteins containing at least twoMycobacterium tuberculosis antigens. In particular, it relates tobi-fusion proteins which contain two individual M. tuberculosisantigens, tri-fusion proteins which contain three M. tuberculosisantigens, tetra-fusion proteins which contain four M. tuberculosisantigens, and penta-fusion proteins which contain five M. tuberculosisantigens, and methods for their use in the diagnosis, treatment andprevention of tuberculosis infection.

2. BACKGROUND OF THE INVENTION

Tuberculosis is a chronic infectious disease caused by infection with M.tuberculosis. It is a major disease in developing countries, as well asan increasing problem in developed areas of the world, with about 8million new cases and 3 million deaths each year. Although the infectionmay be asymptomatic for a considerable period of time, the disease ismost commonly manifested as an acute inflammation of the lungs,resulting in fever and a nonproductive cough. If untreated, seriouscomplications and death typically result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance.

In order to control the spread of tuberculosis, effective vaccinationand accurate early diagnosis of the disease are of utmost importance.Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity. The most common Mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public with this agent.

Diagnosis of tuberculosis is commonly achieved using a skin test, whichinvolves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48-72 hours after injection, whichindicates exposure to Mycobacterial antigens. Sensitivity andspecificity have, however, been a problem with this test, andindividuals vaccinated with BCG cannot be distinguished from infectedindividuals.

While macrophages have been shown to act as the principal effectors ofM. tuberculosis immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection against M.tuberculosis infection is illustrated by the frequent occurrence of M.tuberculosis in Acquired Immunodeficiency Syndrome patients, due to thedepletion of CD4⁺ T cells associated with human immunodeficiency virus(HIV) infection. Mycobacterium-reactive CD4⁺ T cells have been shown tobe potent producers of gamma-interferon (IFN-γ), which, in turn, hasbeen shown to trigger the anti-mycobacterial effects of macrophages inmice. While the role of IFN-γ in humans is less clear, studies haveshown that 1,25-dihydroxy-vitamin D3, either alone or in combinationwith IFN-γ or tumor necrosis factor-alpha, activates human macrophagesto inhibit M. tuberculosis infection. Furthermore, it is known thatIFN-γ stimulates human macrophages to make 1,25-dihydroxy-vitamin D3.Similarly, interleukin-12 (IL-12) has been shown to play a role instimulating resistance to M. tuberculosis infection. For a review of theimmunology of M. tuberculosis infection, see Chan and Kaufmann, 1994,Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASMPress, Washington, D.C.

Accordingly, there is a need for improved vaccines, and improved methodsfor diagnosis, preventing and treating tuberculosis.

3. SUMMARY OF THE INVENTION

The present invention relates to fusion proteins of M. tuberculosisantigens. In particular, it relates to fusion polypeptides that containtwo or more M. tuberculosis antigens, polynucleotides encoding suchpolypeptides, methods of using the polypeptides and polynucleotides inthe diagnosis, treatment and prevention of M. tuberculosis infection.

The present invention is based, in part, on the inventors' discoverythat polynucleotides which contain two to five M. tuberculosis codingsequences produce recombinant fusion proteins that retain theimmunogenicity and antigenicity of their individual components. Thefusion proteins described herein induced both T cell and B cellresponses, as measured by T cell proliferation, cytokine production, andantibody production. Furthermore, a fusion protein was used as animmunogen with adjuvants in vivo to elicit both cell-mediated andhumoral immunity to M. tuberculosis. Additionally, a fusion protein wasmade by a fusion construct and used in a vaccine formulation with anadjuvant to afford long-term protection in animals against thedevelopment of tuberculosis. The fusion protein was a more effectiveimmunogen than a mixture of its individual protein components.

In a specific embodiment of the invention, the isolated or purified M.tuberculosis polypeptides of the invention may be formulated aspharmaceutical compositions for administration into a subject in theprevention and/or treatment of M. tuberculosis infection. Theimmunogenicity of the fusion protein may be enhanced by the inclusion ofan adjuvant.

In another aspect of the invention, the isolated or purifiedpolynucleotides are used to produce recombinant fusion polypeptideantigens in vitro. Alternatively, the polynucleotides may beadministered directly into a subject as DNA vaccines to cause antigenexpression in the subject, and the subsequent induction of an anti-M.tuberculosis immune response.

It is also an object of the invention that the polypeptides be used inin vitro assays for detecting humoral antibodies or cell-mediatedimmunity against M. tuberculosis for diagnosis of infection or monitorof disease progression. Additionally, the polypeptides may be used as anin vivo diagnostic agent in the form of an intradermal skin test.Alternatively, the polypeptides may be used as immunogens to generateanti-M. tuberculosis antibodies in a non-human animal. The antibodiescan be used to detect the target antigens in vivo and in vitro.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. The nucleotide sequence (SEQ ID NO: 1) and amino acidsequence (SEQ ID NO:2) of tri-fusion protein Ra12-TbH9-Ra35 (designatedMtb32-Mtb39 fusion).

FIG. 2: The nucleotide sequence (SEQ ID NO:3) and amino acid sequence(SEQ ID NO:4) of tri-fusion protein Erd14-DPV-MTI.

FIG. 3A-3D: The nucleotide sequence (SEQ ID NO:5) and amino acidsequence (SEQ ID NO:6) of tri-fusion protein TbRa3-38 kD-Tb38-1.

FIG. 4A-4D: The nucleotide sequence (SEQ ID NO:7) and amino acidsequence (SEQ ID NO:8) of bi-fusion protein TbH9-Tb38-1.

FIG. 5A-5J: The nucleotide sequence (SEQ ID NO:9) and amino acidsequence (SEQ ID NO: 10) of tetra-fusion protein TbRa3-38 kD-Tb38-1-DPEP(designated TbF-2).

FIGS. 6A and 6B: The nucleotide sequence (SEQ ID NO:11) and amino acidsequence (SEQ ID NO:12) of penta-fusion protein Erd14-DPV-MTI-MSL-MTCC2(designated Mtb88f).

FIGS. 7A and 7B: The nucleotide sequence (SEQ ID NO: 13) and amino acidsequence (SEQ ID NO:14) of tetra-fusion protein Erd14-DPV-MTI-MSL(designated Mtb46f).

FIGS. 8A and 8B: The nucleotide sequence (SEQ ID NO:15) and amino acidsequence (SEQ ID NO:16) of tetra-fusion protein DPV-MTI-MSL-MTCC2(designated Mtb71 f).

FIGS. 9A and 9B: The nucleotide sequence (SEQ ID NO: 17) and amino acidsequence (SEQ ID NO: 18) of tri-fusion protein DPV-MTI-MSL (designatedMtb31 f).

FIGS. 10A and 10B: The nucleotide sequence (SEQ ID NO:19) and amino acidsequence (SEQ ID NO:20) of tri-fusion protein nTH9-DPV-MTI (designatedMtb61f).

FIG. 11A and II B: The nucleotide sequence (SEQ ID NO:21) and amino acidsequence (SEQ ID NO:22) of tri-fusion protein Erd14-DPV-MTI (designatedMtb36f).

FIGS. 12A and 12B: The nucleotide sequence (SEQ ID NO:23) and amino acidsequence (SEQ ID NO:24) of bi-fusion protein TbH9-Ra35 (designatedMtb59f).

FIGS. 13A and 13B: The nucleotide sequence (SEQ ID NO:25) and amino acidsequence (SEQ ID NO:26) of bi-fusion protein Ra12-DPPD (designatedMtb24).

FIG. 14A-14F: T cell proliferation responses of six PPD+ subjects whenstimulated with two fusion proteins and their individual components.

FIG. 15A-15F: IFN-γ production of six PPD+ subjects when stimulated withtwo fusion proteins and their individual components.

FIG. 16A-16F: T cell proliferation of mice immunized with a fusionprotein or its individual components and an adjuvant.

FIG. 17: IFN-γ production of mice immunized with a fusion protein or itsindividual components and an adjuvant.

FIG. 18: IL4 production of mice immunized with a fusion protein or itsindividual components and an adjuvant.

FIG. 19A-19F: Serum antibody concentrations of mice immunized with afusion protein or its individual components and an adjuvant.

FIG. 20A-20C: Survival of guinea pigs after aerosol challenge of M.tuberculosis. Fusion protein, Mtb32-Mtb39 fusion or a mixture of Mtb32Aand Mtb39A, were formulated in adjuvant SBAS1c (20A), SBAS2 (20B) orSBAS7 (20C), and used as an immunogen in guinea pigs prior to challengewith bacteria. BCG is the positive control.

FIGS. 21A and 21B: Stimulation of proliferation and IFN-γ production inTbH9-specific T cells by the fusion protein TbH9-Tb38-1.

FIGS. 22A and 22B: Stimulation of proliferation and IFN-γ production inTh38-1-specific T cells by the fusion protein TbH9-Tb38-1.

FIGS. 23A and 23B: Stimulation of proliferation and IFN-γ production inT cells previously shown to respond to both TbH-9 and Tb38-1 antigens bythe fusion protein TbH9-Tb38-1.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antigens useful for the treatment andprevention of tuberculosis, polynucleotides encoding such antigens, andmethods for their use. The antigens of the present invention are fusionpolypeptides of M. tuberculosis antigens and variants thereof. Morespecifically, the antigens of the present invention comprise at leasttwo polypeptides of M. tuberculosis that are fused into a larger fusionpolypeptide molecule. The antigens of the present invention may furthercomprise other components designed to enhance the immunogenicity of theantigens or to improve these antigens in other aspects, for example, theisolation of these antigens through addition of a stretch of histidineresidues at one end of the antigen.

5.1. M. tuberculosis Specific Antigens

The antigens of the present invention are exemplified in FIGS. 1Athrough 13B, including homologues and variants of those antigens. Theseantigens may be modified, for example, by adding linker peptidesequences as described below. These linker peptides may be insertedbetween one or more polypeptides which make up each of the fusionproteins presented in FIGS. 1A through 13B. Other antigens of thepresent invention are antigens described in FIGS. 1A through 13B whichhave been linked to a known antigen of M. tuberculosis, such as thepreviously described 38 kD (SEQ ID NO:27) antigen (Andersen and Hansen,1989, Infect. Immun. 57:2481-2488; Genbank Accession No. M30046).

5.2. Immunogenicity Assays

Antigens described herein, and immunogenic portions thereof, have theability to induce an immunogenic response. More specifically, theantigens have the ability to induce proliferation and/or cytokineproduction (i.e., interferon-γ and/or interleukin-12 production) in Tcells, NK cells, B cells and/or macrophages derived from an M.tuberculosis-immune individual. The selection of cell type for use inevaluating an immunogenic response to a antigen will depend-on thedesired response. For example, interleukin-12 production is most readilyevaluated using preparations containing B cells and/or macrophages. AnM. tuberculosis-immune individual is one who is considered to beresistant to the development of tuberculosis by virtue of having mountedan effective T cell response to M. tuberculosis (i.e., substantiallyfree of disease symptoms). Such individuals may be identified based on astrongly positive (i.e., greater than about 10 mm diameter induration)intradermal skin test response to tuberculosis proteins (PPD) and anabsence of any signs or symptoms of tuberculosis disease. T cells, NKcells, B cells and macrophages derived from M. tuberculosis-immuneindividuals may be prepared using methods known to those of ordinaryskill in the art. For example, a preparation of PBMCs (i.e., peripheralblood mononuclear cells) may be employed without further separation ofcomponent cells. PBMCs may generally be prepared, for example, usingdensity centrifugation through “FICOLL” (Winthrop Laboratories, NY). Tcells for use in the assays described herein may also be purifieddirectly from PBMCs. Alternatively, an enriched T cell line reactiveagainst mycobacterial proteins, or T cell clones reactive to individualmycobacterial proteins, may be employed. Such T cell clones may begenerated by, for example, culturing PBMCs from M. tuberculosis-immuneindividuals with mycobacterial proteins for a period of 2-4 weeks. Thisallows expansion of only the mycobacterial protein-specific T cells,resulting in a line composed solely of such cells. These cells may thenbe cloned and tested with individual proteins, using methods known tothose of ordinary skill in the art, to more accurately define individualT cell specificity. In general, antigens that test positive in assaysfor proliferation and/or cytokine production (i.e., interferon-γ and/orinterleukin-12 production) performed using T cells, NK cells, B cellsand/or macrophages derived from an M. tuberculosis-immune individual areconsidered immunogenic. Such assays may be performed, for example, usingthe representative procedures described below. Immunogenic portions ofsuch antigens may be identified using similar assays, and may be presentwithin the polypeptides described herein.

The ability of a polypeptide (e.g., an immunogenic antigen, or a portionor other variant thereof) to induce cell proliferation is evaluated bycontacting the cells (e.g., T cells and/or NK cells) with thepolypeptide and measuring the proliferation of the cells. In general,the amount of polypeptide that is sufficient for evaluation of about 10⁵cells ranges from about 10 ng/ml to about 100 μg/mL and preferably isabout 10 μg/mL. The incubation of polypeptide with cells is typicallyperformed at 37° C. for about six days. Following incubation withpolypeptide, the cells are assayed for a proliferative response, whichmay be evaluated by methods known to those of ordinary skill in the art,such as exposing cells to a pulse of radiolabeled thymidine andmeasuring the incorporation of label into cellular DNA. In general, apolypeptide that results in at least a three fold increase inproliferation above background (i.e., the proliferation observed forcells cultured without polypeptide) is considered to be able to induceproliferation.

The ability of a polypeptide to stimulate the production of interferon-γand/or interleukin-12 in cells may be evaluated by contacting the cellswith the polypeptide and measuring the level of interferon-γ orinterleukin-12 produced by the cells. In general, the amount ofpolypeptide that is sufficient for the evaluation of about 10⁵ cellsranges from about 10 ng/mL to about 100 μg/mL and preferably is about 10μg/mL. The polypeptide may be, but need not be, immobilized on a solidsupport, such as a bead or a biodegradable microsphere, such as thosedescribed in U.S. Pat. Nos. 4,897,268 and 5,075,109. The incubation ofpolypeptide with the cells is typically performed at 37° C. for aboutsix days.

Following incubation with polypeptide, the cells are assayed forinterferon-γ and/or interleukin-12 (or one or more subunits thereof),which may be evaluated by methods known to those of ordinary skill inthe art, such as an enzyme-linked immunosorbent assay (ELISA) or, in thecase of IL-12 P70 subunit, a bioassay such as an assay measuringproliferation of T cells. In general, a polypeptide that results in theproduction of at least 50 pg of interferon-γ per mL of culturedsupernatant (containing 10⁴-10⁵ T cells per mL) is considered able tostimulate the production of interferon-γ. A polypeptide that stimulatesthe production of at least 10 pg/mL of IL-12 P70 subunit, and/or atleast 100 pg/mL of IL-12 P40 subunit, per 10⁵ macrophages or B cells (orper 3×10⁵ PBMC) is considered able to stimulate the production of IL-12.

In general, immunogenic antigens are those antigens that stimulateproliferation and/or cytokine production (i.e., interferon-γ and/orinterleukin-12 production) in T cells, NK cells, B cells and/ormacrophages derived from at least about 25% of M. tuberculosis-immuneindividuals. Among these immunogenic antigens, polypeptides havingsuperior therapeutic properties may be distinguished based on themagnitude of the responses in the above assays and based on thepercentage of individuals for which a response is observed. In addition,antigens having superior therapeutic properties will not stimulateproliferation and/or cytokine production in vitro in cells derived frommore than about 25% of individuals who are not M. tuberculosis-immune,thereby eliminating responses that are not specifically due to M.tuberculosis-responsive cells. Those antigens that induce a response ina high percentage of T cell, NK cell, B cell and/or macrophagepreparations from M. tuberculosis-immune individuals (with a lowincidence of responses in cell preparations from other individuals) havesuperior therapeutic properties.

Antigens with superior therapeutic properties may also be identifiedbased on their ability to diminish the severity of M. tuberculosisinfection in experimental animals, when administered as a vaccine.Suitable vaccine preparations for use on experimental animals aredescribed in detail below. Efficacy may be determined based on theability of the antigen to provide at least about a 50% reduction inbacterial numbers and/or at least about a 40% decrease in mortalityfollowing experimental infection. Suitable experimental animals includemice, guinea pigs and primates.

5.3. Isolation of Coding Sequences

The present invention also relates to nucleic acid molecules that encodefusion polypeptides of M. tuberculosis. In a specific embodiment by wayof example in Section 6, infra, thirteen M. tuberculosis fusion codingsequences were constructed. In accordance with the invention, anynucleotide sequence which encodes the amino acid sequence of the fusionprotein can be used to generate recombinant molecules which direct theexpression of the coding sequence.

In order to clone full-length coding sequences or homologous variants togenerate the fusion polynucleotides, labeled DNA probes designed fromany portion of the nucleotide sequences or their complements disclosedherein may be used to screen a genomic or cDNA library made from variousstrains of M. tuberculosis to identify the coding sequence of eachindividual component. Isolation of coding sequences may also be carriedout by the polymerase chain reactions (PCR) using two degenerateoligonucleotide primer pools designed on the basis of the codingsequences disclosed herein.

The invention also relates to isolated or purified polynucleotidescomplementary to the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23 and 25, and polynucleotides that selectivelyhybridize to such complementary sequences. In a preferred embodiment, apolynucleotide which hybridizes to the sequence of SEQ ID NOS: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 or its complementary sequenceunder conditions of low stringency and encodes a protein that retainsthe immunogenicity of the fusion proteins of SEQ ID NOS:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24 and 26 is provided. By way of example and notlimitation, exemplary conditions of low stringency are as follows (seealso Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792):Filters containing DNA are pretreated for 6 h at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mQ salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe isused. Filters are incubated in hybridization mixture for 18-20 h at 40°C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC,25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another preferred embodiment, a polynucleotide which hybridizes tothe coding sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23 and 25 or its complementary sequence under conditions of highstringency and encodes a protein that retains the immunogenicity of thefusion proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24and 26 is provided. By way of example and not limitation, exemplaryconditions of high stringency are as follows: Prehybridization offilters containing DNA is carried out for 8 h to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/mL denatured salmon sperm DNA.Filters are hybridized for 48 h at 65° C. in prehybridization mixturecontaining 100 μg/mL denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 h in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Thisis followed by a wash in 0.1×SSC at 50° C. for min beforeautoradiography. Other conditions of high stringency which may be usedare well known in the art.

In yet another preferred embodiment, a polynucleotide which hybridizesto the coding sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23 and 25 or its complementary sequence under conditions of moderatestringency and encodes a protein that retains the immunogenicity of thefusion proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24and 26 is provided. Exemplary conditions of moderate stringency are asfollows: Filters containing DNA are pretreated for 6 h at 55° C. in asolution containing 6×SSC, 5× Denhart's solution, 0.5% SDS and 100 μg/mLdenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which may be used are well-known inthe art. Washing of filters is done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.1% SDS.

5.4. Polypeptides Encoded by the Coding Sequences

In accordance with the invention, a polynucleotide of the inventionwhich encodes a fusion protein, fragments thereof, or functionalequivalents thereof may be used to generate recombinant nucleic acidmolecules that direct the expression of the fusion protein, fragmentsthereof, or functional equivalents thereof, in appropriate host cells.The fusion polypeptide products encoded by such polynucleotides may bealtered by molecular manipulation of the coding sequence.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used in the practice of the invention for theexpression of the fusion polypeptides. Such DNA sequences include thosewhich are capable of hybridizing to the coding sequences or theircomplements disclosed herein under low, moderate or high stringencyconditions described in Sections 5.3, supra.

Altered nucleotide sequences which may be used in accordance with theinvention include deletions, additions or substitutions of differentnucleotide residues resulting in a sequence that encodes the same or afunctionally equivalent gene product. The gene product itself maycontain deletions, additions or substitutions of amino acid residues,which result in a silent change thus producing a functionally equivalentantigenic epitope. Such conservative amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine, histidine and arginine; amino acids with uncharged polar headgroups having similar hydrophilicity values include the following:glycine, asparagine, glutamine, serine, threonine and tyrosine; andamino acids with nonpolar head groups include alanine, valine,isoleucine, leucine, phenylalanine, proline, methionine and tryptophan.

The nucleotide sequences of the invention may be engineered in order toalter the fusion protein coding sequence for a variety of ends,including but not limited to, alterations which modify processing andexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, e.g., site-directedmutagenesis, to insert new restriction sites, to alter glycosylationpatterns, phosphorylation, etc.

In an alternate embodiment of the invention, the coding sequence of afusion protein could be synthesized in whole or in part, using chemicalmethods well known in the art. See, e.g., Caruthers et al., 1980, Nuc.Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res.9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.Alternatively, the polypeptide itself could be produced using chemicalmethods to synthesize an amino acid sequence in whole or in part. Forexample, peptides can be synthesized by solid phase techniques, cleavedfrom the resin, and purified by preparative high performance liquidchromatography. (See Creighton, 1983, Proteins Structures And MolecularPrinciples, W.H. Freeman and Co., N.Y. pp. 50-60). The composition ofthe synthetic polypeptides may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton, 1983,Proteins, Structures and Molecular Principles, W.H. Freeman and Co.,N.Y., pp. 34-49).

Additionally, the coding sequence of a fusion protein can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson,C., et al., 1978, J. Biol. Chem. 253:6551), use of TAB® linkers(Pharmacia), and the like. It is important that the manipulations do notdestroy immunogenicity of the fusion polypeptides.

In addition, nonclassical amino acids or chemical amino acid analogs canbe introduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In a specific embodiment, the coding sequences of each antigen in thefusion protein are joined at their amino- or carboxy-terminus via apeptide bond in any order. Alternatively, a peptide linker sequence maybe employed to separate the individual polypeptides that make-up afusion polypeptide by a distance sufficient to ensure that eachpolypeptide folds into a secondary and tertiary structure that maximizesits antigenic effectiveness for preventing and treating tuberculosis.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen-based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may be from 1 to about 50 amino acids in length.Peptide sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference. For example, the antigens in a fusion protein may beconnected by a flexible polylinker such as Gly-Cys-Gly orGly-Gly-Gly-Gly-Ser repeated 1 to 3 times (Bird et al., 1988, Science242:423-426; Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:1066-1070).

In one embodiment, such a protein is produced by recombinant expressionof a nucleic acid encoding the protein. Such a fusion product can bemade by ligating the appropriate nucleic acid sequences encoding thedesired amino acid sequences to each other by methods known in the art,in the proper coding frame, and expressing the product by methods knownin the art. Alternatively, such a product may be made by proteinsynthetic techniques, e.g., by use of a peptide synthesizer. Codingsequences for other molecules such as a cytokine or an adjuvant can beadded to the fusion polynucleotide as well.

5.5. Production of Fusion Proteins

In order to produce a M. tuberculosis fusion protein of the invention,the nucleotide sequence coding for the protein, or a functionalequivalent, is inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. The host cells or celllines transfected or transformed with recombinant expression vectors canbe used for a variety of purposes. These include, but are not limitedto, large scale production of the fusion protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a fusion coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. (See, e.g., the techniquesdescribed in Sambrook et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y.). RNA capable of encoding a polypeptide mayalso be chemically synthesized (Gait, ed, 1984, OligonucleotideSynthesis, IRL Press, Oxford).

5.5.1. Expression Systems

A variety of host-expression vector systems may be utilized to express afusion protein coding sequence. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing a coding sequence; yeast (e.g., Saccharomycdes,Pichia) transformed with recombinant yeast expression vectors containinga coding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing a coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing a coding sequence; or mammalian cell systems (e.g.,COS, CHO, BHK, 293, 3T3 cells). The expression elements of these systemsvary in their strength and specificities.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, may be used in the expression vector. Forexample, when cloning in bacterial systems, inducible promoters such aspL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter;cytomegalovirus promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll α/β, binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter) may be used; when generating cell lines thatcontain multiple copies of a the antigen coding sequence, SV40-, BPV-and EBV-based vectors may be used with an appropriate selectable marker.

Bacterial systems are preferred for the expression of M. tuberculosisantigens. For in vivo delivery, a bacterium such asBacillus-Calmette-Guerrin may be engineered to express a fusionpolypeptide of the invention on its cell surface. A number of otherbacterial expression vectors may be advantageously selected dependingupon the use intended for the expressed products. For example, whenlarge quantities of the fusion protein are to be produced forformulation of pharmaceutical compositions, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which a coding sequence may be ligated into the vector inframe with the lacZ coding region so that a hybrid protein is produced;pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109,Van Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509); and thelike. pGEX vectors may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can be purified easily from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned fusionpolypeptide of interest can be released from the GST moiety.

5.5.2. Protein Purification

Once a recombinant protein is expressed, it can be identified by assaysbased on the physical or functional properties of the product, includingradioactive labeling of the product followed by analysis by gelelectrophoresis, radioimmunoassay, ELISA, bioassays, etc.

Once the encoded protein is identified, it may be isolated and purifiedby standard methods including chromatography (e.g., high performanceliquid chromatography, ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. The actualconditions used will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art. The functional properties may be evaluatedusing any suitable assay such as antibody binding, induction of T cellproliferation, stimulation of cytokine production such as IL2, IL4 andIFN-γ. For the practice of the present invention, it is preferred thateach fusion protein is at least 80% purified from other proteins. It ismore preferred that they are at least 90% purified. For in vivoadministration, it is preferred that the proteins are greater than 95%purified.

5.6. Uses of the Fusion Protein Coding Sequence

The fusion protein coding sequence of the invention may be used toencode a protein product for use as an immunogen to induce and/orenhance immune responses to M. tuberculosis. In addition, such codingsequence may be ligated with a coding sequence of another molecule suchas cytokine or an adjuvant. Such polynucleotides may be used in vivo asa DNA vaccine (U.S. Pat. Nos. 5,589,466; 5,679,647; 5,703,055). In thisembodiment of the invention, the polynucleotide expresses its encodedprotein in a recipient to directly induce an immune response. Thepolynucleotide may be injected into a naive subject to prime an immuneresponse to its encoded product, or administered to an infected orimmunized subject to enhance the secondary immune responses.

In a preferred embodiment, a therapeutic composition comprises a fusionprotein coding sequence or fragments thereof that is part of anexpression vector. In particular, such a polynucleotide contains apromoter operably linked to the coding region, said promoter beinginducible or constitutive, and, optionally, tissue-specific. In anotherembodiment, a polynucleotide contains a coding sequence flanked byregions that promote homologous recombination at a desired site in thegenome, thus providing for intrachromosomal expression of the codingsequence (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe subject. These two approaches are known, respectively, as in vivo orex vivo gene transfer.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded fusion proteinproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing it as part of an appropriate nucleic acidexpression vector and administering it so that it becomes intracellular,e.g., by infection using a defective or attenuated retroviral or otherviral vector (see, U.S. Pat. No. 4,980,286), or by direct injection ofnaked DNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules (U.S. Pat. Nos. 5,407,609; 5,853,763; 5,814,344 and5,820,883), or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) which can be used to target cell typesspecifically expressing the receptors, etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992; WO 92/22635 dated Dec. 23,1992; WO92/20316 dated Nov. 26, 1992; WO93/14188 dated Jul. 22, 1993; WO93/20221 dated Oct. 14, 1993). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector such as a retroviral vector canbe used (see, Miller et al., 1993, Meth. Enzymol. 217:581-599).Retroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. A fusion coding sequence is cloned into the vector,which facilitates delivery of the nucleic acid into a recipient. Moredetail about retroviral vectors can be found in Boesen et al., 1994,Biotherapy 6:291-302, which describes the use of a retroviral vector todeliver the mdr1 gene to hematopoietic stem cells in order to make thestem cells more resistant to chemotherapy. Other references illustratingthe use of retroviral vectors in gene therapy are: Clowes et al., 1994,J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossmanand Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Adeno-associated virus (AAV)has also been proposed for use in in vivo gene transfer (Walsh et al.,1993, Proc. Soc. Exp. Biol. Med. 204:289-300.

Another approach involves transferring a construct to cells in tissueculture by such methods as electroporation, lipofection, calciumphosphate mediated transfection, or viral infection. Usually, the methodof transfer includes the transfer of a selectable marker to the cells.The cells are then placed under selection to isolate those cells thathave taken up and are expressing the transferred gene. Those cells arethen delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention.

The polynucleotides of the invention may also be used in the diagnosisof tuberculosis for detection of polynucleotide sequences specific to M.tuberculosis in a patient. Such detection may be accomplished, forexample, by isolating polynucleotides from a biological sample obtainedfrom a patient suspected of being infected with the bacteria. Uponisolation of polynucleotides from the biological sample, a labeledpolynucleotide of the invention that is complementary to one or more ofthe polynucleotides, will be allowed to hybridize to polynucleotides inthe biological sample using techniques of nucleic acid hybridizationknown to those of ordinary skill in the art.

For example, such hybridization may be carried out in solution or withone hybridization partner on a solid support.

5.7. Therapeutic and Prophylactic Uses of the Fusion Protein

Purified or partially purified fusion proteins or fragments thereof maybe formulated as a vaccine or therapeutic composition. Such compositionmay include adjuvants to enhance immune responses. In addition, suchproteins may be further suspended in an oil emulsion to cause a slowerrelease of the proteins in vivo upon injection. The optimal ratios ofeach component in the formulation may be determined by techniques wellknown to those skilled in the art.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to enhance the immune response. Most adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a specific or nonspecificstimulator of immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis. Suitable adjuvants are commerciallyavailable and include, for example, Freund's Incomplete Adjuvant andFreund's Complete Adjuvant (Difco Laboratories) and Merck Adjuvant 65(Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvantsinclude alum, biodegradable microspheres, monophosphoryl lipid A, quilA, SBAS1c, SBAS2 (Ling et al., 1997, Vaccine 15:1562-1567), SBAS7,Al(OH)₃ and CpG oligonucleotide (WO96/02555).

In the vaccines of the present invention, it is preferred that theadjuvant induces an immune response comprising Th1 aspects. Suitableadjuvant systems include, for example, a combination of monophosphoryllipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL)together with an aluminum salt. An enhanced system involves thecombination of a monophosphoryl lipid A and a saponin derivative,particularly the combination of 3D-MLP and the saponin QS21 as disclosedin WO 94/00153, or a less reactogenic composition where the QS21 isquenched with cholesterol as disclosed in WO 96/33739. Previousexperiments have demonstrated a clear synergistic effect of combinationsof 3D-MLP and QS21 in the induction of both humoral and Th1 typecellular immune responses. A particularly potent adjuvant formationinvolving QS21, 3D-MLP and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210 and is a preferred formulation.

Formulations containing an antigen of the present invention may beadministered to a subject per se or in the form of a pharmaceutical ortherapeutic composition. Pharmaceutical compositions comprising theproteins may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the polypeptides into preparations whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

For topical administration, the proteins may be formulated as solutions,gels, ointments, creams, suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the proteins may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the proteins may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, a composition can be readily formulated bycombining the proteins with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the proteins to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, such aslactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the proteins may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the proteins for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the proteins and a suitable powder base suchas lactose or starch.

The proteins may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the proteins mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theproteins may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Alternatively, other pharmaceutical deliverysystems may be employed. Liposomes and emulsions are well known examplesof delivery vehicles that may be used to deliver an antigen. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. The fusion proteinsmay also be encapsulated in microspheres (U.S. Pat. Nos. 5,407,609;5,853,763; 5,814,344 and 5,820,883). Additionally, the proteins may bedelivered using a sustained-release system, such as semipermeablematrices of solid polymers containing the therapeutic or vaccinatingagent. Various sustained-release materials have been established and arewell known by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the proteins for a few weeksup to over 100 days. Depending on the chemical nature and the biologicalstability of the reagent, additional strategies for proteinstabilization may be employed.

Determination of an effective amount of the fusion protein for inducingan immune response in a subject is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immune response using techniques that are well known inthe art. One having ordinary skill in the art could readily optimizeadministration to humans based on animal data. Dosage amount andinterval may be adjusted individually. For example, when used as avaccine, the polypeptides and/or polynucleotides of the invention may beadministered in about 1 to 3 doses for a 1-36 week period. Preferably, 3doses are administered, at intervals of about 3-4 months, and boostervaccinations may be given periodically thereafter. Alternate protocolsmay be appropriate for individual patients. A suitable dose is an amountof polypeptide or DNA that, when administered as described above, iscapable of raising an immune response in an immuunized patientsufficient to protect the patient from M. tuberculosis infection for atleast 1-2 years. In general, the amount of polypeptide present in a dose(or produced in situ by the DNA in a dose) ranges from about 1 pg toabout 100 mg per kg of host, typically from about 10 pg to about 1 mg,and preferably from about 100 pg to about 1 μg. Suitable dose range willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

5.8 Diagnostic Uses of the Fusion Protein

The fusion polypeptides of the invention are useful in the diagnosis oftuberculosis infection in vitro and in vivo. The ability of apolypeptide of the invention to induce cell proliferation or cytokineproduction can be assayed by the methods disclosed in Section 5.2,Supra.

In another aspect, this invention provides methods for using one or moreof the fusion polypeptides to diagnose tuberculosis using a skin test invivo. As used herein, a skin test is any assay performed directly on apatient in which a delayed-type hypersensitivity (DTH) reaction (such asswelling, reddening or dermatitis) is measured following intradermalinjection of one or more polypeptides as described above. Such injectionmay be achieved using any suitable device sufficient to contact thepolypeptide with dermal cells of the patient, such as, for example, atuberculin syringe or 1 mL syringe. Preferably, the reaction is measuredat least about 48 hours after injection, more preferably about 48 toabout 72 hours after injection.

The DTH reaction is a cell-mediated immune response, which is greater inpatients that have been exposed previously to the test antigen (i.e.,the immunogenic portion of the polypeptide employed, or a variantthereof). The response may be measured visually, using a ruler. Ingeneral, a response that is greater than about 0.5 cm in diameter,preferably greater than about 1.0 cm in diameter, is a positiveresponse, indicative of tuberculosis infection, which may or may not bemanifested as an active disease.

The fusion polypeptides of this invention are preferably formulated, foruse in a skin test, as pharmaceutical compositions containing apolypeptide and a physiologically acceptable carrier. Such compositionstypically contain one or more of the above polypeptides in an amountranging from about 1 μg to about 100 μg, preferably from about 10 μg toabout 50 μg in a volume of 0.1 mL. Preferably, the carrier employed insuch pharmaceutical compositions is a saline solution with appropriatepreservatives, such as phenol and/or Tween 80™.

In another aspect, the present invention provides methods for using thepolypeptides to diagnose tuberculosis. In this aspect. methods areprovided for detecting M. tuberculosis infection in a biological sampleusing the fusion polypeptides alone or in combination. As used herein, a“biological sample” is any antibody-containing sample obtained from apatient. Preferably, the sample is whole blood, sputum, serum, plasma,saliva cerebrospinal fluid or urine. More preferably, the sample is ablood, serum or plasma sample obtained from a patient or a blood supply.The polypeptide(s) are used in an assay, as described below, todetermine the presence or absence of antibodies to the polypeptide(s) inthe sample relative to a predetermined cut-off value. The presence ofsuch antibodies indicates previous sensitization to mycobacterialantigens which may be indicative of tuberculosis.

In embodiments in which more than one fusion polypeptide is employed,the polypeptides used are preferably complementary (i.e., one componentpolypeptide will tend to detect infection in samples where the infectionwould not be detected by another component polypeptide). Complementarypolypeptides may generally be identified by using each polypeptideindividually to evaluate serum samples obtained from a series ofpatients known to be infected with M. tuberculosis. After determiningwhich samples test positive (as described below) with each polypeptide,combinations of two or more fusion polypeptides may be formulated thatare capable of detecting infection in most, or all, of the samplestested. Such polypeptides are complementary. Approximately 25-30% ofsera from tuberculosis-infected individuals are negative for antibodiesto any single protein. Complementary polypeptides may, therefore, beused in combination to improve sensitivity 3 of a diagnostic test.

There are a variety of assay formats known to those of ordinary skill inthe art for using one or more polypeptides to detect antibodies in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988, which is incorporated herein byreference. In a preferred embodiment, the assay involves the use ofpolypeptide immobilized on a solid support to bind to and remove theantibody from the sample. The bound antibody may then be detected usinga detection reagent that contains a reporter group. Suitable detectionreagents include antibodies that bind to the antibody/polypeptidecomplex and free polypeptide labeled with a reporter group (e.g., in asemi-competitive assay). Alternatively, a competitive assay may beutilized, in which an antibody that binds to the polypeptide is labeledwith a reporter group and allowed to bind to the immobilized antigenafter incubation of the antigen with the sample. The extent to whichcomponents of the sample inhibit the binding of the labeled antibody tothe polypeptide is indicative of the reactivity of the sample with theimmobilized polypeptide.

The solid support may be any solid material known to those of ordinaryskill in the art to which the antigen may be attached. For example. thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example. in U.S. Pat. No. 5,359,681.

The polypeptides may be bound to the solid support using a variety oftechniques known to those of ordinary skill in the art. In the contextof the present invention, the term “bound” refers to both noncovalentassociation, such as adsorption, and covalent attachment (which may be adirect linkage between the antigen and functional groups on the supportor may be a linkage by way of a cross-linking agent). Binding byadsorption to a well in a microtiter plate or to a membrane ispreferred. In such cases. adsorption may be achieved by contacting thepolypeptide, in a suitable buffer, with the solid support for a suitableamount of time. The contact time varies with temperature, but istypically between about 1 hour and 1 day. In general, contacting a wellof a plastic microtiter plate (such as polystyrene or polyvinylchloride)with an amount of polypeptide ranging from about 10 ng to about 1 Mg,and preferably about 100 ng, is sufficient to bind an adequate amount ofantigen. Covalent attachment of polypeptide to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the polypeptide. For example, thepolypeptide may be bound to supports having an appropriate polymercoating using benzoquinone or by condensation of an aldehyde group onthe support with an amine and an active hydrogen on the polypeptide(see, e.g., Pierce Immunotechnology Catalog and Handbook. 1991, atA12-A13).

In certain embodiments. the assay is an enzyme linked immunosorbent 1assay (ELISA). This assay may be performed by first contacting a fusionpolypeptide antigen that has been immobilized on a solid support,commonly the well of a microtiter plate, with the sample, such thatantibodies to the polypeptide within the sample are allowed to bind tothe immobilized polypeptide. Unbound sample is then removed from theimmobilized polypeptide and a detection reagent capable of binding tothe immobilized antibody-polypeptide complex is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific detectionreagent.

More specifically, once the polypeptide is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.) may be employed. The immobilizedpolypeptide is then incubated with the sample, and antibody is allowedto bind to the antigen. The sample may be diluted with a suitablediluent, such as phosphate-buffered saline (PBS) prior to incubation. Ingeneral, an appropriate contact time is that period of time that issufficient to detect the presence of antibody within a M.tuberculosis-infected sample. Preferably, the contact time is sufficientto achieve a level of binding that is at least 95% of that achieved atequilibrium between bound and unbound antibody. Those of ordinary skillin the art will recognize that the time necessary to achieve equilibriummay be readily determined by assaying the level of binding that occursover a period of time. At room temperature, an incubation time of about30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. Detectionreagent may then be added to the solid support. An appropriate detectionreagent is any compound that binds to the immobilizedantibody-polypeptide complex and that can be detected by any of avariety of means known to those in the art. Preferably, the detectionreagent contains a binding agent (for example, Protein A, Protein G,lectin or free antigen) conjugated to a reporter group. Preferredreporter groups include enzymes (such as horseradish peroxidase),substrates, cofactors, inhibitors, dyes, radionuclides, luminescentgroups, fluorescent groups, biotin and colloidal particles, such ascolloidal gold and selenium. The conjugation of binding agent toreporter group may be achieved using standard methods known to those ofordinary skill in the art. Common binding agents may also be purchasedconjugated to a variety of reporter groups from many commercial sources(e.g. Zymed Laboratories, San Francisco, Calif., and Pierce, Rockford.Ill.).

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound antibody. An appropriate amount of time may generally bedetermined from the manufacturer's instructions or by assaying the levelof binding that occurs over a period of time. Unbound detection reagentis then removed and bound detection reagent is detected using thereporter group. The method employed for detecting the reporter groupdepends upon the nature of the reporter group. For radioactive groups,scintillation counting or autoradiographic methods are generallyappropriate. Spectroscopic methods may be used to detect dyes,luminescent groups and fluorescent groups. Biotin may be detected usingavidin, coupled to a different reporter group (commonly a radioactive orfluorescent group or an enzyme). Enzyme reporter groups may generally bedetected by the addition of substrate (generally for a specific periodof time). followed by spectroscopic or other analysis of the reactionproducts.

To determine the presence or absence of anti-M. tuberculosis antibodiesin the sample, the signal detected from the reporter group that remainsbound to the solid support is generally compared to a signal thatcorresponds to a predetermined cut-off value. In one preferredembodiment, the cut-off value is the average mean signal obtained whenthe immobilized antigen is incubated with samples from an uninfectedpatient. In general, a sample generating a signal that is three standarddeviations above the predetermined cut-off value is considered positivefor tuberculosis. In an alternate preferred embodiment, the cut-offvalue is determined using a Receiver Operator Curve. according to themethod of Sackett et al., 1985, Clinical Epidemiology: A Basic Sciencefor Clinical Medicine, Little Brown and Co., pp. 106-107. Briefly, inthis embodiment, the cut-off value may be determined from a plot ofpairs of true positive rates (i.e., sensitivity) and false positiverates (100%-specificity) that correspond to each possible cut-off valuefor the diagnostic test result. The cut-off value on the plot that isthe closest to the upper left-hand corner (i.e. the value that enclosesthe largest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate. Ingeneral, a sample generating a signal that is higher than the cut-offvalue determined by this method is considered positive for tuberculosis.

In a related embodiment, the assay is performed in a rapid flow-throughor strip test format, wherein the antigen is immobilized on a membrane,such as nitrocellulose. In the flow-through test, antibodies within thesample bind to the immobilized polypeptide as the sample passes throughthe membrane. A detection reagent (e.g., protein A-colloidal gold) thenbinds to the antibody-polypeptide complex as the solution containing thedetection reagent flows through the membrane. The detection of bounddetection reagent may then be performed as described above. In the striptest format, one end of the membrane to which polypeptide is bound isimmersed in a solution containing the sample. The sample migrates alongthe membrane through a region containing detection reagent and to thearea of immobilized polypeptide. Concentration of detection reagent atthe polypeptide indicates the presence of anti-M. tuberculosisantibodies in the sample. Typically, the concentration of detectionreagent at that site generates a pattern, such as a line, that can beread visually. The absence of such a pattern indicates a negativeresult. In general, the amount of polypeptide immobilized on themembrane is selected to generate a visually discernible pattern when thebiological sample contains a level of antibodies that would besufficient to generate a positive signal in an ELISA, as discussedabove. Preferably, the amount of polypeptide immobilized on the membraneranges from about 5 ng to about 1 μg, and more preferably from about 50ng to about 500 ng. Such tests can typically be performed with a verysmall amount (e.g., one drop) of patient serum or blood.

The invention having been described, ihe following examples are offeredby way of illustration and not limitation.

6. EXAMPLE Fusion Proteins of M. tuberculosis Antigens RetainImmunogenicity of the Individual Components

6.1. Materials and Methods

6.1.1. Construction of Fusion Proteins

Coding sequences of M. tuberculosis antigens were modified by PCR inorder to facilitate their fusion and subsequent expression of fusionprotein. DNA amplification was performed using 10 μl 10×Pfu buffer, 2 μl10 mM dNTPs, 2 μl each of the PCR primers at 10 μM concentration, 81.5μl water, 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and 1μl DNA at either 70 ng/μl (for ThRa3 antigen) or 50 ng/μl (for 38 kD andTb38-1 antigens). For TbRa3 antigen, denaturation at 94° C. wasperformed for 2 min, followed by 40 cycles of 96° C. for 15 sec and 72°C. for 1 min, and lastly by 72° C. for 4 min. For 38 kD antigen,denaturation at 96° C. was performed for 2 min, followed by 40 cycles of96° C. for 30 sec, 68° C. for 15 sec and 72° C. for 3 min and finally by72° C. for 4 min. For Tb38-1 antigen, denaturation at 94° C. for 2 minwas followed by 10 cycles of 96° C. for 15 sec, 68° C. for 15 sec and72° C. for 1.5 min, 30 cycles of 96° C. for 15 sec, 64° C. for 15 secand 72° C. for 1.5, and finally by 72° C. for 4 min. Following digestionwith a restriction endonuclease to yield the desired cohesive or bluntends, a polynucleotide specific for each fusion polypeptide was ligatedinto an expression plasmid. Each resulting plasmid contained the codingsequences of the individual antigens of each fusion polypeptide. Theexpression vectors used were pET-12b and pT7̂L2 IL 1.

Three coding sequences for antigens Ra12, TbH9 and Ra35 were ligated toencode one fusion protein (SEQ ID NOS: 1 and 2) (FIGS. 1A and 2B).Another three coding sequences for antigens Erd14, DPV and MTI wereligated to encode a second fusion protein (SEQ ID NOS:3 and 4) (FIG. 2).Three coding sequences for antigens TbRa3, 38 kD and Tb38-1 were ligatedto encode one fusion protein (SEQ ID NOS:5 and 6) (FIG. 3A-3D). Twocoding sequences for antigens TbH9 and Tb38-1 were ligated to encode onefusion protein (SEQ ID NOS:7 and 8) (FIG. 4A-4D). Four coding sequencesfor antigens ThRa3, 38 kD, Tb38-1 and DPEP were ligated to encode onefusion protein (SEQ ID NOS:9 and 10) (FIG. 5A-5J). Five coding sequencesfor antigens Erd14, DPV, MTI, MSL and MTCC2 were ligated to encode onefusion protein (SEQ ID NOS: 11 and 12) (FIGS. 6A and 6B). Four codingsequences for antigens Erd14, DPV, MTI and MSL were ligated to encodeone fusion protein (SEQ ID NOS:13 and 14) (FIGS. 7A and 7B). Four codingsequences for antigens DPV, MTI, MSL and MTCC2 were ligated to encodeone fusion protein (SEQ ID NOS: 15 and 16) (FIGS. 8A and 8B). Threecoding sequences for antigens DPV, M11 and MSL were ligated to encodeone fusion protein (SEQ ID NOS:17 and 18) (FIGS. 9A and 9B). Threecoding sequences for antigens TbH9, DPV and MTI were ligated to encodeone fusion protein (SEQ ID NOS:19 and 20) (FIGS. 10A and 10B). Threecoding sequences for antigens Erd14, DPV and MTI were ligated to encodeone fusion protein (SEQ ID NOS:21 and 22) (FIGS. 11A and 11B). Twocoding sequences for antigens TbH9 and Ra35 were ligated to encode onefusion protein (SEQ ID NOS:23 and 24) (FIGS. 12A and 12B). Two codingsequences for antigens Ra12 and DPPD were ligated to encode one fusionprotein (SEQ ID NOS:25 and 26) (FIGS. 13A and 13B).

The recombinant proteins were expressed in E. coli with six histidineresidues at the amino-terminal portion using the pET plasmid vector(pET-17b) and a T7 RNA polymerase expression system (Novagen, Madison,Wis.). E. coli strain BL21 (DE3) pLysE (Novagen) was used for high levelexpression. The recombinant (His-Tag) fusion proteins were purified fromthe soluble supernatant or the insoluble inclusion body of 500 ml ofIPTG induced batch cultures by affinity chromatography using the onestep QIAexpress Ni-NTA Agarose matrix (QIAGEN, Chatsworth, Calif.) inthe presence of 8M urea. Briefly, 20 ml of an overnight saturatedculture of BL21 containing the pET construct was added into 500 ml of2xYT media containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol,grown at 37° C. with shaking. The bacterial cultures were induced with 2mM IPTG at an OD 560 of 0.3 and grown for an additional 3 h (OD=1.3 to1.9). Cells were harvested from 500 ml batch cultures by centrifugationand resuspended in 20 ml of binding buffer (0.1 M sodium phosphate, pH8.0; 10 mM Tris-HCl, pH 8.0) containing 2 mM PMSF and 20 μg/ml leupeptinplus one complete protease inhibitor tablet (Boehringer Mannheim) per 25ml. E. coli was lysed by freeze-thaw followed by brief sonication, thenspun at 12 k rpm for 30 min to pellet the inclusion bodies.

The inclusion bodies were washed three times in 1% CHAPS in 10 mMTris-HCl (pH 8.0). This step greatly reduced the level of contaminatingLPS. The inclusion body was finally solubilized in 20 ml of bindingbuffer containing 8 M urea or 8M urea was added directly into thesoluble supernatant. Recombinant fusion proteins with His-Tag residueswere batch bound to Ni-NTA agarose resin (5 ml resin per 500 mlinductions) by rocking at room temperature for 1 h and the complexpassed over a column. The flow through was passed twice over the samecolumn and the column washed three times with 30 ml each of wash buffer(0.1 M sodium phosphate and 10 mM Tris-HCL, pH 6.3) also containing 8 Murea. Bound protein was eluted with 30 ml of 150 mM immidazole in washbuffer and 5 ml fractions collected. Fractions containing eachrecombinant fusion protein were pooled, dialyzed against 10 mM TrisHCl(pH 8.0) bound one more time to the Ni-NTA matrix, eluted and dialyzedin 10 mM Tris-HCL (pH 7.8). The yield of recombinant protein varies from25-150 mg per liter of induced bacterial culture with greater than 98%purity. Recombinant proteins were assayed for endotoxin contaminationusing the Limulus assay (BioWhittaker) and were shown to contain<10E.U.Img.

6.1.2. T-Cell Proliferation Assay

Purified fusion polypeptides were tested for the ability to induceT-cell proliferation in peripheral blood mononuclear cell (PBMC)preparations. The PBMCs from donors known to be PPD skin test positiveand whose T-cells were shown to proliferate in response to PPD and crudesoluble proteins from M. tuberculosis were cultured in RPMI 1640supplemented with 10% pooled human serum and 50 μg/ml gentamicin.Purified polypeptides were added in duplicate at concentrations of 0.5to 10 μg/ml. After six days of culture in 96-well round-bottom plates ina volume of 200 μlt, 50 μl of medium was removed from each well fordetermination of IFN-γ levels, as described below in Section 6.1.3. Theplates were then pulsed with 1 μCi/well of tritiated thymidine for afurther 18 hours, harvested and tritium uptake determined using a gasscintillation counter. Fractions that resulted in proliferation in bothreplicates three fold greater than the proliferation observed in cellscultured in medium alone were considered positive.

6.1.3. Interferon-γ Assay

Spleens from mice were removed asceptically and single cell suspensionprepared in complete RPMI following lysis of red blood cells. 100 μl ofcells (2×10⁻⁵ cells) were plated per well in a 96-well flat bottommicrotiter plate. Cultures were stimulated with the indicatedrecombinant proteins for 24 h and the supernatant assayed for IFN-γ.

The levels of supernatant IFN-γ was analysed by sandwich ELISA, usingantibody pairs and procedures available from PharMingen. Standard curveswere generated using recombinant mouse cytokines. ELISA plates (Corning)were coated with 50 μl/well (1 μg/ml, in 0.1 M bicarbonate coatingbuffer, pH9.6) of a cytokine capture mAb (rat anti-mouse IFN-γ(PharMingen; Cat. # 18181 D)), and incubated for 4 h at room temp. Shakeout plate contents and block with PBS-0.05% Tween, 1.0% BSA (200μl/well) overnight at 4° C. and washed for 6× in PBS-0.1% Tween.Standards (mouse IFN-γ) and supernatant samples diluted in PBS-0.05%Tween, 0.1% BSA were then added for 2 hr at room temp. The plates werewashed as above and then incubated for 2 hr at room temperature with 100μl/well of a second Ab (biotin rat a mouse IFN-γ (Cat. # 18112D;PharMingen) at 0.5 μg/ml diluted in PBS-0.05% Tween, 0.1% BSA. Afterwashing, plates were incubated with 100 μl/well of streptavidin-HRP(Zymed) at a 1:2500 dilution in PBS-0.05% Tween, 0.1% BSA at room tempfor 1 hr. The plates were washed one last time and developed with 100μl/well TMB substrate (3,3′,5,5′-tetramethylbenzidine, Kirkegaard andPerry, Gaithersburg, Md.) and the reaction stopped after colordeveloped, with H₂SO₄, 50 μl/well. Absorbance (OD) were determined at450 nm using 570 nm as a reference wavelength and the cytokineconcentration evaluated using the standard curve.

6.2. Results

6.2.1. Tri-Fusion Proteins Induced Immune Responses

Three coding sequences for M. tuberculosis antigens were inserted intoan expression vector for the production of a fusion protein. Theantigens designated Ra12, TbH9 and Ra35 were produced as one recombinantfusion protein (FIGS. 1A and 1B).

Antigens Erd14, DPV and MTI were produced as a second fusion protein(FIG. 2). The two fusion proteins were affinity purified for use in invitro and in vivo assays.

The two fusion proteins were tested for their ability to stimulate Tcell responses from six PPD⁺ subjects. When T cell proliferation wasmeasured, both fusion proteins exhibited a similar reactivity pattern astheir individual components (FIG. 14A-14F). A similar result wasobtained when IFN-γ production was measured (FIG. 15A-15F). For example,subject D160 responded to antigens TbH9 and MTI individually. SubjectD160 also responded to the fusion proteins that contained these antigens(FIGS. 14B and 15B).

In contrast, no T cell response from D160 was observed to other antigensindividually.

Another subject, D201, who did not react with antigens Erd14, DPV or MTIindividually, was also unresponsive to the fusion protein containingthese antigens. It should be noted that when the T cell responses to theindividual components of the two fusion proteins were not particularlystrong, the fusion proteins stimulated responses that were equal to orhigher than that induced by the individual antigens in most cases.

The Ra12-TbH9-Ra35 tri-fusion protein was also tested as an immunogen invivo.

In these experiments, the fusion protein was injected into the footpadsof mice for immunization. Each group of three mice received the proteinin a different adjuvant formulation: SBAS1c, SBAS2 (Ling et al., 1997,Vaccine 15:1562-1567), SBAS7 and AL(OH)₃. After two subcutaneousimmunizations at three week intervals, the animals were sacrificed oneweek later, and their draining lymph nodes were harvested for use asresponder cells in T cell proliferation and cytokine production assays.Regardless which adjuvant was used in the immunization, strong T cellproliferation responses were induced against TbH9 when it was used as anindividual antigen (FIG. 16A). Weaker responses were induced againstRa35 and Ra12 (FIGS. 16B and 16C). When the Ra12-TbH9-Ra35 fusionprotein was used as immunogen, a response similar to that against theindividual components was observed.

When cytokine production was measured, adjuvants SBAS1c and SBAS2produced similar IFN-γ (FIG. 17) and ILK responses (FIG. 18). However,the combination of SBAS7 and aluminum hydroxide produced the strongestIFN-γ responses and the lowest level of IL-4 production for all threeantigens. With respect to the humoral antibody response in vivo, FIG.19A-19F shows that the fusion protein elicited both IgG₁ and IgG_(2a)antigen-specific responses when it was used with any of the threeadjuvants.

Additionally, C57BL/6 mice were immunized with an expression constructcontaining Ra12-TbH9-Ra35 (Mtb32-Mtb39 fusion) coding sequence as DNAvaccine. The immunized animals exhibited significant protection againsttuberculosis upon a subsequent aerosol challenge of live bacteria. Basedon these results, a fusion construct of Mtb32-Mtb39 coding sequence wasmade, and its encoded product tested in a guinea pig long termprotection model. In these studies, guinea pigs were immunized with asingle recombinant fusion protein or a mixture of Mtb32A (Ra35) andMtb39A (TbH9) proteins in formulations containing an adjuvant. FIG.20A-20C shows that guinea pigs immunized with the fusion protein inSBAS1c or SBAS2 were better protected against the development oftuberculosis upon subsequent challenge, as compared to animals immunizedwith the two antigens in a mixture in the same adjuvant formulation. Thefusion proteins in SBAS2 formulation afforded the greatest protection inthe animals. Thus, fusion proteins of various M. tuberculosis antigensmay be used as more effective immunogens in vaccine formulations than amixture of the individual components.

6.2.2. Bi-Fusion Protein Induced Immune Responses

A bi-fusion fusion protein containing the TbH-9 and Tb38-1 antigenswithout a hinge sequence was produced by recombinant methods. Theability of the TbH9-Tb38-1 fusion protein to induce T cell proliferationand IFN-γ production was examined. PBMC from three donors were employed:one donor had been previously shown to respond to TbH9 but not to Tb38-1(donor 131); one had been shown to respond to Tb38-1 but not to TbH9(donor 184); and one had been shown to respond to both antigens (donor201). The results of these studies demonstrate the functional activityof both the antigens in the fusion protein (FIGS. 21A and 21B, 22A and22B, and 23A and 23B).

6.2.3. A Tetra-Fusion Protein Reacted with Tuberculosis Patient Sera

A fusion protein containing TbRa3, 38 KD antigen, Tb38-1 and DPEP wasproduced by recombinant methods. The reactivity of this tetra-fusionprotein referred to as TbF-2 with sera from M. tuberculosis-infectedpatients was examined by ELISA. The results of these studies (Table 1)demonstrate that all four antigens function independently in the fusionprotein.

One of skill in the art will appreciate that the order of the individualantigens within each fusion protein may be changed and that comparableactivity would be expected provided that each of the epitopes is stillfunctionally available. In addition, truncated forms of the proteinscontaining active epitopes may be used in the construction of fusionproteins.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, nucleotide or amino acid sequences which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims. It is also tobe understood that all base pair sizes given for nucleotides areapproximate and are used for purposes of description.

All publications cited herein are incorporated by reference in theirentirety.

TABLE 1 REACTIVITY OF TBF-2 FUSION PROTEIN WITH TB AND NORMAL SERA TbFTbF-2 ELISA Reactivity Serum ID Status OD450 Status OD450 Status 38 kDTbRa3 Tb38-I DPEP B931-40  TB 0.57 + 0.321 + − + − + B931-41  TB 0.601 +0.396 + + + + − B931-109 TB 0.494 + 0.404 + + + ±± − B931-132 TB 1.502 +1.292 + + + + ±± 5004 TB 1.806 + 1.666 + ±± ±± + − 15004 TB 2.862 +2.468 + + + + − 39004 TB 2.443 + 1.722 + + + + − 68004 TB 2.871 +2.575 + + + + − 99004 TB 0.691 + 0.971 + − ±± + − 107004 TB 0.875 +0.732 + − ±± + − 92004 TB 1.632 + 1.394 + + ±± ±± − 97004 TB 1.491 +1.979 + + ±± − + 118004 TB 3.182 + 3.045 + + ±± − − 173004 TB 3.644 +3.578 + + + + − 175004 TB 3.332 + 2.916 + + + − − 274004 TB 3.696 +3.716 + − + − + 276004 TB 3.243 + 2.56 + − − + − 282004 TB 1.249 +1.234 + + − − − 289004 TB 1.373 + 1.17 + − + − − 308004 TB 3.708 +3.355 + − − + − 314004 TB 1.663 + 1.399 + − − + − 317004 TB 1.163 +0.92 + + − − − 312004 TB 1.709 + 1.453 + − + − − 380004 TB 0.238 −0.461 + − ±± − + 451004 TB 0.18 − 0.2 − − − − ±± 478004 TB 0.188 −0.469 + − − − ±± 410004 TB 0.384 + 2.392 + ±± − − + 411004 TB 0.306 +0.874 + − + − + 421004 TB 0.357 + 1.456 + − + − + 528004 TB 0.047 −0.196 − − − − + A6-87 Normal 0.094 − 0.063 − − − − − A6-88 Normal 0.214− 0.19 − − − − − A6-89 Normal 0.248 − 0.125 − − − − − A6-90 Normal 0.179− 0.206 − − − − − A6-91 Normal 0.135 − 0.151 − − − − − A6-92 Normal0.064 − 0.097 − − − − − A6-93 Normal 0.072 − 0.098 − − − − − A6-94Normal 0.072 − 0.064 − − − − − A6-95 Normal 0.125 − 0.159 − − − − −A6-96 Normal 0.121 − 0.12 − − − − − Cut-off 0.284 0.266

1-13. (canceled)
 14. A polynucleotide comprising a nucleotide sequenceencoding a fusion protein comprising: (i) a DPV antigen consisting ofresidues 9 to 90 of SEQ ID NO:16, or an immunogenic fragment thereof;(ii) a MTI antigen consisting of residues 93 to 186 of SEQ ID NO: 16, oran immunogenic fragment thereof; (iii) a MSL antigen consisting ofresidues 189 to 285 of SEQ ID NO: 16, or an immunogenic fragmentthereof; and (iv) a MTCC2 antigen consisting of residues 288 to 710 ofSEQ ID NO:16, or an immunogenic fragment thereof.
 15. The polynucleotideof claim 14, comprising a nucleotide sequence encoding a fusion proteincomprising: (i) a DPV antigen consisting of residues 9 to 90 of SEQ IDNO: 16; (ii) a MTI antigen consisting of residues 93 to 186 of SEQ IDNO: 16; (iii) a MSL antigen consisting of residues 189 to 285 of SEQ IDNO: 16; and (iv) a MTCC2 antigen consisting of residues 288 to 710 ofSEQ ID NO:
 16. 16. The polynucleotide of claim 14, comprising anucleotide sequence encoding a fusion protein consisting of: (i) a DPVantigen consisting of residues 9 to 90 of SEQ ID NO: 16, or animmunogenic fragment thereof; (ii) a MTI antigen consisting of residues93 to 186 of SEQ ID NO: 16, or an immunogenic fragment thereof; (iii) aMSL antigen consisting of residues 189 to 285 of SEQ ID NO: 16, or animmunogenic fragment thereof; and (iv) a MTCC2 antigen consisting ofresidues 288 to 710 of SEQ ID NO:16, or an immunogenic fragment thereof.17. The polynucleotide of claim 16, comprising a nucleotide sequenceencoding a fusion protein consisting of: (i) a DPV antigen consisting ofresidues 9 to 90 of SEQ ID NO: 16; (ii) a MTI antigen consisting ofresidues 93 to 186 of SEQ ID NO: 16; (iii) a MSL antigen consisting ofresidues 189 to 285 of SEQ ID NO:16; and (iv) a MTCC2 antigen consistingof residues 288 to 710 of SEQ ID NO:16.
 18. The polynucleotide of claim14, consisting of a nucleotide sequence encoding a fusion proteinconsisting of: (i) a DPV antigen consisting of residues 9 to 90 of SEQID NO: 16, or an immunogenic fragment thereof; (ii) a MTI antigenconsisting of residues 93 to 186 of SEQ ID NO: 16, or an immunogenicfragment thereof; (iii) a MSL antigen consisting of residues 189 to 285of SEQ ID NO: 16, or an immunogenic fragment thereof; and (iv) a MTCC2antigen consisting of residues 288 to 710 of SEQ ID NO: 16, or animmunogenic fragment thereof.
 19. The polynucleotide of claim 18,comprising a nucleotide sequence encoding a fusion protein consistingof: (i) a DPV antigen consisting of residues 9 to 90 of SEQ ID NO: 16;(ii) a MTI antigen consisting of residues 93 to 186 of SEQ ID NO: 16;(iii) a MSL antigen consisting of residues 189 to 285 of SEQ ID NO: 16;and (iv) a MTCC2 antigen consisting of residues 288 to 710 of SEQ ID NO:16.
 20. The polynucleotide of claim 14, wherein the antigens orimmunogenic fragments thereof in the encoded fusion are joined at theamino- or carboxy-termini via peptide bonds.
 21. The polynucleotide ofclaim 14, wherein the antigens or immunogenic fragments thereof in theencoded fusion are joined via peptide linkers from 1 to about 50 aminoacids in length.
 22. The polynucleotide of claim 14, comprising anucleotide sequence encoding a fusion protein comprising residues 9 to710 of SEQ ID NO:16.
 23. The polynucleotide of claim 22, comprising anucleotide sequence encoding a fusion protein comprising residues 3 to710 of SEQ ID NO:16.
 24. The polynucleotide of claim 14, comprising anucleotide sequence encoding a fusion protein consisting of residues 9to 710 of SEQ ID NO:
 16. 25. The polynucleotide of claim 14, comprisinga nucleotide sequence encoding a fusion protein consisting of residues 3to 710 of SEQ ID NO:
 16. 26. The polynucleotide of claim 14, consistingof a nucleotide sequence encoding a fusion protein consisting ofresidues 9 to 710 of SEQ ID NO:
 16. 27. The polynucleotide of claim 14,consisting of a nucleotide sequence encoding a fusion protein consistingof residues 3 to 710 of SEQ ID NO:
 16. 28. The polynucleotide of claim14, which is in an expression vector.
 29. The polynucleotide of claim22, which is in an expression vector.
 30. An isolated host celltransfected with the polynucleotide of claim
 28. 31. The host cell ofclaim 30, wherein the host cell is a Bacillus-Calmette-Guerrin.
 32. Apharmaceutical composition comprising the polynucleotide of claim 14.33. The pharmaceutical composition of claim 32, wherein thepolynucleotide comprises a nucleotide sequence encoding a fusion proteincomprising residues 9 to 710 of SEQ ID NO:16.
 34. A method for thetreatment and/or prevention of tuberculosis comprising administering aneffective amount of the polynucleotide of claim
 14. 35. The method ofclaim 34, wherein the polynucleotide comprises a nucleotide sequenceencoding a fusion protein comprising residues 9 to 710 of SEQ ID NO: 16.36. A method of recombinantly making a fusion protein, comprising thestep of expressing the polynucleotide of claim 14 in a host cell.